Apical-basal polarity of the spectrin cytoskeleton in the C. elegans vulva

The C. elegans vulva is a polarized epithelial tube that has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis. Here we used endogenous fusions to show that the spectrin cytoskeleton is polarized in this organ, with conventional beta-spectrin ( UNC-70 ) found only at basolateral membranes and beta heavy spectrin ( SMA-1 ) found only at apical membranes. The sole alpha-spectrin ( SPC-1 ) is present at both locations but requires SMA-1 for its apical localization. Thus, beta spectrins are excellent markers for vulva cell membranes and polarity.

. Apical-basal polarity of the spectrin cytoskeleton in the C. elegans vulva: 6/14/2023 -Open Access A, A') SPC-1/α-spectrin marks all vulva cell membranes; A' is a cartoon tracing of the 7 vulA-vulF rings. B, B') UNC-70/βspectrin marks basolateral membranes. C, C') SMA-1/βH-spectrin marks apical cell membranes. B' and C' cartoons are drawn in comparison to A'. D) VAB-10a/spectraplakin marks apical vulva cell membranes. E, F) SMA-1/βH-spectrin is required for apical localization of SPC-1/α-spectrin (E) but not VAB-10a/spectraplakin (F). F' shows DIC channel, with disorganized tissue occluding the connection between the gonad and the dorsal apex of the vulva (cog phenotype, arrow). All images are single confocal Z-slices through the medial portion of the mid-L4 vulva tube and are representative of at least n=10 animals examined per genotype.
In D. melanogaster, the two β-spectrins exhibit apical-basal polarity in epithelial cells, with conventional β-spectrin found primarily at basolateral membranes and βH-spectrin found primarily at apical membranes, while α-spectrin is found at both locations (Thomas and Kiehart 1994;Thomas and Williams 1999). D. melanogaster βH-spectrin can also influence apical localization of other factors (Zarnescu and Thomas 1999;Dubreuil et al. 2000;Pogodalla et al. 2021). In C. elegans, immunolocalization and/or transgenic reporter studies in the embryo detected UNC-70/β-spectrin at many sites of cell-cell contact but SMA-1/βH-spectrin only at apical membranes (Moorthy et al. 2000;Norman and Moerman 2002;Praitis et al. 2005), suggesting that apical vs. basal partitioning of different β-spectrins is conserved; however, there has been limited analysis of spectrin polarity in larval or adult epithelia (Wirshing and Cram 2018).
Here we visualized the spectrin cytoskeleton in the C. elegans vulva. The vulva is a polarized epithelial tube used for egglaying. It has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis (Sharma-Kishore et al. 1999;Schindler and Sherwood 2013;Gauthier and Rocheleau 2017). Vulva anatomy is well characterized and apical vs. basal cell surfaces can be distinguished easily using simple light microscopy of live animals.
The similarly polarized distributions of β-spectrin and βH-spectrin in C. elegans and D. melanogaster suggest this may be an ancestral feature of metazoan β-spectrins that has been conserved across the many hundreds of millions of years since the evolutionary divergence of nematodes and insects. Some vertebrate tissues also exhibit a polarized spectrin cytoskeleton, but the clear distinction between conventional β-spectrins and βH spectrin appears to have been lost in vertebrates, since either can be found apically or basolaterally (Stabach and Morrow 2000;Cortese et al. 2017;Mylvaganam et al. 2020Mylvaganam et al. , 2022. The roles of polarized spectrins in vulva development remain to be determined. Vulva cell types must divide in a stereotyped fashion, migrate inward to form a luminal cavity, and then assemble into doughnut-like rings via cell-cell fusion (Sulston and Horvitz 1977;Sharma-Kishore et al. 1999). These rings adopt specific shapes and undergo stereotypical movements during morphogenesis. The vulva also must connect appropriately with the uterus to allow the passage of eggs, and with the sex muscles and neurons that control egg-laying (Trent et al. 1983;Schindler and Sherwood 2013). Many of these cell behaviors depend on the actin cytoskeleton and/or on interactions with apical or basal extracellular matrices (Bulik and Robbins 2002;Farooqui et al. 2012;Hagedorn et al. 2013;Morrissey et al. 2014;Yang et al. 2017;Cohen et al. 2020). We observed a variety of vulva shape abnormalities in sma-1 mutants (e.g. Fig. 1F'), which may involve some of the above processes and will be described in a separate report. In the meantime, the spectrin fusions described here will be useful membrane markers for studies of vulva development.

Methods
Caenorhabditis elegans strains were grown at 20˚C under standard conditions (Brenner 1974). For immobilization during imaging, L4 larvae were mounted on 2% agar pads containing 20 mM sodium azide, along with 10 mM levamisole in a drop of M9 buffer. Confocal z-stacks were collected with a 63X Plan Apo objective (HC PL APO CS2 63x/1.30 GLYC) on a Leica TCS SP8 confocal microscope. Images were processed in FIJI (Schindelin et al. 2012) and the panels assembled with Adobe Illustrator.
CRISPR/Cas9-mediated genome editing to tag endogenous SMA-1 with GFP was performed by Suny Biotech. The tag is inserted at the SMA-1 C-terminus, immediately preceding the stop codon, as shown below. This endogenous fusion is functional based on normal body morphology, brood size and embryonic viability of the homozygotes.